Ethanol inhibits the function of ATP-gated membrane ion channels type 4 (P2X4 receptors) in various types of neurons and it has been suggested that P2X4 receptors may be involved in the mechanisms of alcohol actions in the CNS. However, the mechanism of alcohol modulation of P2X4 receptors function has not been determined. We investigated ethanol inhibition of the rat P2X4 receptor and the contribution of the three histidine residues in the extracellular loop of this receptor to ethanol inhibition of receptor function, using site-directed mutagenesis and electrophysiological characterization of recombinant receptors. In the wildtype receptor, 50, 200 and 500 mM ethanol increasingly shifted the ATP concentration-response curve to the right in a parallel manner, increasing the EC50 value without affecting Emax. However, 750 or 900 mM ethanol did not produce a further increase in the EC50 value of the ATP concentration-response curve, suggesting that this inhibition is not competitive. The P2X4 receptor mutations H140A and H286A did not significantly alter ethanol inhibition of ATP-activated current. By contrast, the mutation H241A changed the mechanism by which ethanol inhibits receptor function; viz., ethanol inhibition was not associated with an increased EC50 value of the ATP concentration-response curve, instead, ethanol decreased the maximal response to ATP by without affecting the EC50 value of the ATP concentration-response curve. Ethanol inhibition of the H241A mutant was voltage independent between -60 and +20 mV and ethanol did not alter the reversal potential of ATP-activated current. In addition, ethanol decreased the desensitization rate of the H241A-mediated current. The purinoceptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2?,4?-disulphonic acid (PPADS), did not alter the magnitude of ethanol inhibition of ATP-activated current in the H241A mutant. The results suggest that ethanol inhibits the wildtype rat P2X4 receptor by an allosteric action to increase the EC50 value of the ATP concentration-response curve, the P2X4 receptor mutation H241A alters the mechanism by which ethanol inhibits P2X4 receptor function, and ethanol and PPADS or suramin appear to inhibit H241A-mutated receptors at independent sites. Experiments are also in progress to elucidate the cellular mechanisms of alcohol and neuroactive substance action on neurotransmitter receptors such as NMDA receptors, GABA-A receptors, nicotinic acetylcholine (nACh) receptors and serotonin type 3 (5-HT3) receptors. These studies hold the promise that such physiological approaches will advance our knowledge of the cellular mechanisms of alcohol and neuroactive substance action in the nervous system and provide a foundation for understanding the cellular basis of alcohol abuse and alcoholism.